A multiplex analysis method allows the simultaneous detection of the potential presence of several analytes within a same sample. A multiplex analysis method is typically implemented using a solid support comprising spots, for example a microplate comprising spots in each well, the spots each being intended to detect an analyte or to serve as a control.
It is clear for one skilled in the art that one risk related to spot technology is the absence of deposit, the elimination or deterioration of several spots during the preparation method for the solid support, in particular a microplate, or during the implementation of the analysis method using said solid support. The device for depositing samples or reagents may indeed accidentally come into contact with one or several spots, thereby altering their surface, for example by forming a striation in one or several spots, or by pulling out all or part of one or more spots.
For example, in the article by Bastarache et al. (Accuracy and Reproducibility of a multiplex Immunoassay platform: a validation study, J. Immunol Methods, Mar. 31, 2011, 367 (1-2) 33-39), flaws are presented that were observed at the end of testing on the light signal, such as spot irregularities, the presence of comas that cause the contamination of one spot by a neighboring spot and the absence of expected signal at the theoretical location of the spot.
Yet when a negative result is rendered at the end of the analysis method, this result must result from the absence of the analyte to be detected in the sample, and not from an absence or a deterioration of the detection spot of the corresponding analyte. Securing analysis methods is crucial, in particular for their use in diagnostics in humans, for example to verify the absence of viral or bacterial contamination of a blood sample for transfusion purposes.
The manufacture of a solid support for a spot analysis consists of depositing, on the surface of the solid support, solutions comprising a capture ligand of the analyte to be detected, so as to form spots. The quality of the solid support is next verified at the end of the manufacturing of the solid support, so as to keep only the solid supports having intact and well-formed spots.
Thus, document US2006/0063197 describes the use of a fluorophore in the deposition solution intended to form the spots of a DNA microarray. The fluorophore is used to verify the quality of each spot at the end of the preparation method for the DNA microarray. Furthermore, only a weak residual fluorescence signal is detected at the spots before adding the substrate, during the implementation of an ELISA test.
Document WO2012/142397 examines flux issues in microfluidic apparatuses containing DNA microarrays and describes a DNA microarray assembly comprising an array chamber with an inlet for the sample at a first end, a DNA microarray and an outlet for the sample at a second end connected to a waste chamber, the area transverse to the first end of the array chamber being wider than that at the second end. This document also describes a method for verifying the manufacturing quality of a DNA microarray, by measuring the fluorescence emitted by an internal quality control fluorophore at the spots of an array and encoding the information relative to each spot of the array in a barcode, a memory device or by radiofrequency identification, this information thus encoded being associated with the DNA microarray.
Furthermore, during the implementation of a multiplex analysis method, the signal corresponding to the analyte to be detected can be detected at a theoretical reading grid. This theoretical reading grid is generally defined at the end of the multiplex analysis method, from the signal detected at a spot serving as a positive control. However, in light of the reading scales, any shift in the positioning of the spots during the manufacture of the solid support and/or in the positioning of the solid support in the device for detecting the signal relative to the theoretical reading grid causes a shift in the actual positioning of the spots relative to the theoretical reading grid and therefore affects the detection sensitivity of the analytes.
There is therefore still a need for solutions to secure the results obtained at the end of an analysis method on spots, in particular by making it possible to verify the presence, location and/or integrity of the spots at the end of the analysis method and/or by making it possible to optimize the detection of analytes, for example by improving the detection sensitivity of the analytes.